1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for the introduction of DNA using circular molecules that are not able to replicate outside a host cell genome.
2. General Description of the Related Art
The production of genetically modified organisms involves the introduction of DNA sequences into, or in addition to, the existing genome of a recipient cell, tissue or organism. This transformation of new genetic material has been reported for a wide variety of organisms including, but not limited to, yeast, bacteria, mammals, plants, viruses and insects. In each case, specialized vectors are required for the proper insertion, maintenance or expression of the introduced DNA.
DNA used for transformation can be either double- or single-stranded, and can be circular or linear in form. In a laboratory, DNA is commonly used for transformation in its double-stranded (ds) form, although DNA in single-stranded form (ssDNA) has been used to transform yeast (see Gietz and Woods, 2001). In nature, single-stranded (ss) DNA intermediates are involved during transformation by Agrobacterium tumefaciens and some viruses. Circular, double-stranded DNA (dsDNA) is the most commonly used conformation for transformation (Sambrook and Russell, 2001; Ausubel et al., 2001) although linear ds-DNA may be used as well, for example, for transformation of yeast (Raymond et al., 1999) or microprojectile bombardment of plant cells (see, for example, U.S. Pat. Nos. 6,153,811 and 6,0404,97). Generation of transformable DNA requires that one of ordinary skill in the art perform several operations including, but not limited to, ligation of the proper DNA pieces needed for maintenance of the vector and expression of the desired genes, passage of the completed vector through the proper host cells to increase the number of molecules, purification of the vector in the desired form for transformation and, if using linear DNA molecules, preparation and purification of the linear fragment from a circular molecule or other starting nucleic acid source.
Currently, circular dsDNA molecules are propagated by passage through a bacterial host and typically contain bacterial origins of replication or other associated sequences. It is often undesirable to have “ancillary sequences,” such as bacterial origins of replication, in the transformed organism. It would be advantageous to have a means of producing circular DNA molecules that do not contain ancillary sequences, such as origins of replication, in ample numbers for transformation. Circular molecules without ancillary sequences would not require removal of these ancillary sequences in the target host or removal prior to the transformation of the host.
There is a need to simplify the preparation of circular, dsDNA molecules for transformation without the need for ancillary sequences usually associated with maintenance of exogenous sequences in cells typically used for vector production. Furthermore, a method is needed to produce sufficient quantities of circular DNAs for use in direct DNA delivery transformation methods.